<strong>Context:</strong> High vitamin D and physical activity (PA) levels are independently associated with improved body composition and muscle function in older adults.<p></p> <p><strong>Objective:</strong> To investigate the interaction of 25-hydroxyvitamin D (25OHD) and PA status in maintenance of body composition and muscle function in older adults.</p> <p><strong>Design and Setting:</strong> A five-year prospective population-based study of Australian community-dwelling older adults.</p> <p><strong>Participants:</strong> 615 community-dwelling volunteers aged ≥ 50 years (61.4 ± 6.9 [mean ± SD] years; 48% female) randomly selected from electoral rolls and categorised according to baseline serum 25OHD (≥ or < 50nmol/L) and PA (≥ or < 10,000 pedometer-determined steps/day) levels as: high 25OHD and high PA (VitD+PA+); high 25OHD and low PA (VitD+PA-); low 25OHD and high PA (VitD-PA+); low 25OHD and low PA (VitD-PA-). A subset of 518 participants completed accelerometer assessments during follow-up.</p> <p><strong>Main Outcome Measures:</strong> Changes in DXA-assessed body composition and lower-limb muscle function.</p> <p><strong>Results:</strong> VitD+PA+ had significantly smaller increases in body fat over five years compared to other groups (all P<i>P</i> < 0.05). Higher baseline pedometer-determined PA resulted in declines in total body fat (β = -0.23kg per 100 steps/day, <i>P</i> = 0.001) over five years for participants with high 25OHD, but not those with low 25OHD (<i>P</i> > 0.05). Amongst participants with accelerometer data, these associations were generally mediated by higher levels of moderate/vigorous PA.</p> <p><strong>Conclusions:</strong> High vitamin D status appears to enhance PA-related declines in body fat during ageing but the mechanism may be greater amounts of outdoor moderate/vigorous PA rather than a direct effect of 25OHD.</p>